靶向肿瘤相关巨噬细胞的pH-还原双敏感MicroRNA载体及其抗肿瘤免疫研究

Tumor-associated macrophages (TAMs)，which are the predominant infiltrated leukocytes in solid tumors, play an important role in promoting tumor angiogenesis and metastasis as well as immune escape. Repolarization of TAMs to tumor-suppressive M1 type macrophages has been considered as a promising strategy for cancer therapy. In the current project, we propose a TAM-targeted pH-reduction dual-sensitive polypeptide micelles as miRNA vector, which will deliver miRNA into TAMs, and promote their differentiation to M1 macrophages by regulating gene expression. Polypeptide copolymers will be first synthesized by ring opening polymerization of N-carboxyanhydride, followed by self-assembly in reductive/oxidative conditions to generate dual-responsive micelles. The effect of peptide sequence and polymerization as well as redox-sensitivity of micelles on transfection efficacy and TAM differentiation will be determined in vitro, and the most effective micelles with minial cytotoxicity will be selected for the future study. The micelles will then be modified with TAM-specific ligands in order to enhance the uptake of miRNA by TAMs. Moreover, the micelles will also be covered with pH-sensitive sheddable PEG to prevent non-specific capture and "off target effect " of miRNA in vivo. The anti-cancer immune responses induced by micelle-formulated miRNA will be evaluated in tumor-bearing mice. The results of current project will not only contribute to the development of miRNA delivery systems, but also demonstrate TAM-targeted miRNA delivery as a promising therapeutic strategy for anti-cancer treatment.

肿瘤相关巨噬细胞（TAM）作为肿瘤组织中主要的侵润性白细胞,在肿瘤细胞的免疫逃逸和转移等多个过程中扮演重要角色,是肿瘤免疫治疗的重要靶点。对此,本项目将构建一种靶向TAM的pH-还原双敏感聚多肽纳米胶束作为miRNA载体，通过调控TAM的基因表达，诱导其分化成为具有"抑癌"作用的M1型巨噬细胞，从而有效地引发抗肿瘤免疫反应。拟深入研究纳米胶束的理化性质对miRNA基因调控作用的影响和内在规律；综合调控胶束的氨基酸序列以及还原响应性，增强miRNA对TAM的诱导分化；采用被动和主动协同靶向方式提高TAM对miRNA的摄取；同时结合pH敏感的可脱落PEG修饰，防止miRNA的"脱靶效应"。本项目研究成果将不但为研制高效安全的miRNA载体供理论依据，还将为肿瘤免疫治疗提供新策略。

本项目拟构建靶向肿瘤相关巨噬细胞（tumor-associated macrophage, TAM）的聚多肽纳米胶束作为miRNA载体；通过综合调控纳米颗粒的氨基酸序列和还原响应性，增强miRNA对TAM的基因调控作用；采用被动+主动联合靶向方式提高miRNA的TAM靶向性；结合pH敏感的PEG修饰，防止miRNA的“脱靶效应”；旨在诱导TAM分化成为具有抑癌作用的M1型活化巨噬细胞，从而引发抗肿瘤免疫反应。.本项目在执行期内，按照合同规定组织实施项目研制工作。主要研究内容包括：（1）研制了靶向TAM的pH-还原双敏感聚多肽纳米载体（sPEG/GLC），采用sPEG/GLC 负载miRNA-155（miR-155），促使肿瘤组织中的TAM分化成为具有抗肿瘤作用的M1型巨噬细胞，抑制肿瘤生长。（2）研制了靶向TAM的锌卟啉-聚多肽纳米胶束（Znpp-LEG），探讨Znpp-LEG负载免疫佐剂polyI:C（PIC）对TAM的活化作用和分子机制。结果表明，Znpp-LEG/P纳米颗粒通过上调TAM胞内ROS，下调免疫抑制性STAT3信号，有效地增强PIC对TAM的活化作用，从而抑制肿瘤生长。（3）采用聚多肽纳米疫苗联合负载免疫佐剂（poly I:C）和miR-148a抑制剂，通过调控肿瘤相关DC（TADC）中失调的miR-148/DNMT1/SOCS1轴，促使TADC成为成熟活化的DC，从而有效地克服肿瘤微环境中的免疫耐受，增强机体的抗肿瘤免疫应答。研究成果为提高肿瘤免疫治疗的临床疗效提供了理论基础和创新策略。相关结果以通讯作者发表SCI论文10篇，其中影响因子〉5的论文9篇；在国内/国际重大学术会议发表会议论文6篇，口头报告/邀请报告5次，墙报交流9次；申请国内发明专利2项；培养博士研究生1名，硕士研究生3名。